Insulin is a polypeptide hormone secreted by beta-cells of the pancreas. This hormone is made up of two polypeptide chains, an A-chain of 21 amino acids, and a B-chain of 30 amino acids. These two chains are linked to one another in the mature form of the hormone by two interchain disulfide bridges. The A-chain also features one intra-chain disulfide bridge.
Insulin is a hormone that is synthesized in the body in the form of a single-chain precursor molecule, proinsulin. Proinsulin is a molecule comprised of a prepeptide of 24 amino acids, followed by the B-chain peptide, a C-peptide of 35 amino acids, and an A-chain peptide. The C-peptide of this precursor insulin molecule contains the two amino acids, lysine-arginine (LR) at its carboxy end (where it attaches to the A-chain), and the two amino acids, arginine-arginine (RR) at its amino end (where it attaches to the B-chain). In the mature insulin molecule, the C-peptide is cleaved away from the peptide so as to leave the A-chain and the B-chain connected directly to one another in its active form.
Molecular biology techniques have been used to produce human proinsulin. In this regard, three major methods have been used for the production of this molecule. Two of these methods involve Escherichia coli, with either the expression of a large fusion protein in the cytoplasm (Chance et al. (1981), and Frank et al. (1981) in Peptides: Proceedings of the 7th American Peptide Chemistry Symposium (Rich, D. and Gross, E., eds.), pp. 721-728, 729-739, respectively, Pierce Chemical Company, Rockford, Ill.), or the use of a signal peptide to enable secretion into the periplasmic space (Chan et al. (1981) P.N.A.S., U.S.A., 78:5401-5404). A third method utilizes yeast, especially Saccharomyces cerevisiae, to secrete the insulin precursor into the medium (Thim, et al. (1986), P.N.A.S., U.S.A., 83: 6766-6770).
Chance et al. report a process for preparing insulin by producing each of the A and B chains of insulin in the form of a fusion protein by culturing E. coli that carries a vector compromising a DNA encoding the fusion protein, cleaving the fusion protein with cyanogen bromide to obtain the A and the B chains, sulfonating the A and B chains to obtain sulfonated chains, reacting the sulfonated B chain with an excess amount of the sulfonated A chain; and then purifying the resultant products to obtain insulin. Drawbacks associated with this process are that it requires two fermentation processes and the requirement of a reaction step for preparing the sulfonated A chain and the sulfonated B chain. This results in a low insulin yield.
Frank et al. described a process for preparing insulin in the form of a fusion protein in E. coli. In this process, proinsulin is produced in the form of a fusion protein by culturing E. coli which carries a vector comprising a nucleic acid sequence (DNA) encoding for the fusion protein, cutting the fusion protein with cyanogens bromide to obtain proinsulin, sulfonating the proinsulin and separation of the sulfonated proinsulin, refolding the sulfonated proinsulin to form correct disulfide bonds, treating the refolded proinsulin with trypsin and carboxypeptidase B, and then purifying the resultant product to obtain insulin. However, the yield of the refolded proinsulin having correctly-folded disulfide bonds is reported to sharply decrease as the concentration of the proinsulin increases. This is allegedly due to, at least among other reasons, misfolding of the protein, and some degree of polymerization being involved. Hence, the process entails the inconvenience of using laborious purification steps during the recovery of proinsulin and consequently any final insulin product.
Thim et al. report a process for producing insulin in yeast, Saccharomyces cerevisiae. This process has the steps of producing a single chain insulin analog having a certain amino acid sequence by culturing Saccharomyces cerevisiae cells, and isolating insulin therefrom through the steps of: purification, enzyme reaction, acid hydrolysis and a second purification. This process, however, results in an unacceptably low yield of insulin.
The role of the native C-peptide in the folding of proinsulin is not precisely known. The dibasic terminal amino acid sequence at both ends of the C-peptide sequence has been considered necessary to preserve the proper processing and/or folding of the proinsulin molecule to insulin. For example, U.S. Pat. No. 5,962,267 describes dibasic terminal amino acid sequences at both ends of the C-peptide. However, modification and/or deletion of other amino acids within the C-peptide sequence has been reported.
For example, Chang et al. (1998) (Biochem. J., 329:631-635) described a shortened C-peptide of a five (5) amino acid length, -YPGDV- (SEQ ID NO: 1), that includes a preserved terminal di-basic amino acid sequence, RR at one terminal end, and LR at the other terminal end, of the peptide. Preservation of the dibasic amino acid residues at the B-chain-C peptide (B-C) and C-peptide-A-chain junctures is taught as being a minimal requirement for retaining the capacity for converting the proinsulin molecule into a properly folded mature insulin protein. The production of the recombinant human insulin was described using E. coli with a shortened C-peptide having a dibasic amino acid terminal sequence.
U.S. Pat. No. 7,087,408 also describes insulin precursors and insulin precursor analogs having a mini C-peptide comprising at least one aromatic amino acid residue. However, cleavage of the mini C-peptide from the B chain may be enabled by cleavage at the natural Lys(B29) amino acid residue in the B chain giving rise to a des-Thr(B30) insulin precursor or analogs thereof.
One of the difficulties and/or inefficiencies associated with the production of recombinant insulin employing a proinsulin construct having the conserved, terminal di-basic amino acid sequence in the C-peptide region is the presence of impurities, such as Arg-(A(0))-insulin, in the reaction mixture, once enzymatic cleavage to remove the C-peptide is performed. This occurs as a result of misdirected cleavage of the proinsulin molecule so as to cleave the C-peptide sequence away from the A-chain at this juncture, by the action of trypsin. Trypsin is a typical serine protease, and hydrolyses a protein or peptide at the carboxyl terminal of an arginine or lysine residue (Enzymes, pp. 261-262 (1979), ed. Dixon, M. & Webb, E. C. Longman Group Ltd., London). This unwanted hydrolysis results in the unwanted Arg(A(0))-insulin by-product, and typically constitutes about 10% of the reaction yield. Hence, an additional purification step is required. The necessity of an additional purification step makes the process much more time consuming, and thus expensive, to use. Moreover, an additional loss of yield may be expected from the necessity of this additional purification step.
Others have described the use of proinsulin constructs that do not have a conserved terminal dibasic amino acid sequence of the C-peptide region. For example, U.S. Pat. No. 6,777,207 (Kjeldsen et al.) relates to a novel proinsulin peptide construct containing a shortened C-peptide that Includes the two terminal amino acids, glycine-arginine or glycine-lysine at the carboxyl terminal end that connects to the A-chain of the peptide. The B-chain of the proinsulin construct described therein has a length of 29 amino acids, in contrast to the native 30 amino acid length of the native B-chain in human insulin. The potential effects of this change to the native amino acid sequence of the B-chain in the human insulin produced are yet unknown. Methods of producing insulin using these proinsulin constructs in yeast are also described. Inefficiencies associated with correct folding of the mature insulin molecule when yeast utilized as the expression host, render this process, among other things, inefficient and more expensive and time consuming to use. In addition, yeast provides a relatively low insulin yield, due to the intrinsically low expression levels of a yeast system as compared to E. coli. 
An ongoing difficulty with this conversion methodology has been and continues to be the presence of substantially large amounts of difficultly-removable by-products in the reaction mixture. Enzymatic modification of human proinsulin using trypsin and carboxypeptidase B results in accumulation of insulin derivatives, leading to more complicated purification processes. Specifically, in the conversion of human proinsulin to human insulin, a large amount (about 4-6%) of desthreonine (des-Thr(B30)) human insulin is formed. Des-Thr(B30) human insulin differs from human insulin by the absence of a single terminal amino acid and requires difficult and cumbersome purification methods to remove. U.S. Pat. No. 5,457,066 describes treating human insulin precursor with trypsin and carboxypeptidase B in an aqueous medium containing about 0.1 to about 2 moles of metal ions (specifically nickel ions), per mole of human insulin precursor. However, the use of metal ions as described in this patent may lead to potential production problems, among other concerns.
Son, et al., “Effects of citraconylation on enzymatic modification of human proinsulin using trypsin and carboxypeptidase B.,” Biotechnol Prog. 25(4) (July-August 2009):1064-70, describes citraconylation and decitraconylation in the enzymatic modification process to reduce des-Thr(B30) human insulin formation.
Many of the foregoing technical problems are equally applicable to the production of insulin analogs. Insulin analogs are altered forms of native insulin that are available to the body for performing the same action as native insulin. One particular insulin analog known as glargine insulin has been described, e.g., in U.S. Pat. Nos. 5,547,930, 5,618,913, and 5,834,422. This analog is used in the treatment of diabetes. Glargine insulin is characterized as a slow release insulin analog that controls blood sugar when no food is being digested. Glargine insulin may form a hexamer when injected subcutaneously into the patient. This insulin analog has been available commercially as LANTUS® (SANOFI AVENTIS®). LANTUS® is an insulin analog wherein the molecule includes a Gly(A21)-Arg(B31)-Arg(B32) amino acid sequence.
Another particular insulin analog known as aspart insulin has been described, e.g., in U.S. Pat. Nos. 5,618,913, 5,547,930, and 5,834,422. This analog is also used in the treatment of diabetes. Aspart insulin analog has increased charge repulsion as compared with native insulin, which prevents the formation of hexamers and thus results in a faster acting insulin. This aspart insulin analog has been available commercially as NOVOLOG® (ELI LILLY®). NOVOLOG® is an insulin analog wherein the molecule includes a Asp(B28) amino acid sequence in place of the native insulin Pro(B28). NOVOLOG® is an injectable, fast-acting insulin. NOVOLOG® is also available as mix with insulin aspart protamine and commercially referred to as NOVOLOG® Mix 70/30, which contains 30% insulin aspart and 70% insulin aspart protamine. The insulin aspart protamine portion is a crystalline form of insulin aspart, which delays the action of the insulin, giving NOVOLOG® Mix 70/30 a prolonged absorption profile after injection.
Another particular insulin analog known as Lis-Pro insulin has been described, e.g., in U.S. Pat. Nos. 5,474,978 and 5,504,188. This analog is used in the treatment of diabetes. Lis-Pro insulin is characterized as a short acting insulin analog, which, when combined with an insulin pump, allows for better blood glucose stability without the risk of hyperglycemia. This Lis-Pro insulin analog has been available commercially as HUMALOG® (ELI LILLY®). HUMALOG® is an insulin analog wherein the molecule includes a Lys(B28)-Pro(B29) amino acid sequence in place of the native insulin Pro(B28)-Lys(B29). HUMALOG® is an injectable, fast-acting insulin.
Accordingly, a need exists for a more efficient process for production of human insulin that is efficient, eliminates currently necessary purification steps, and that at the same time improves and/or preserves acceptable production yield requirements of the pharmaceutical industry.